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      Silk genes and silk gene expression in the spider Tengella perfuga (Zoropsidae), including a potential cribellar spidroin (CrSp)

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          Abstract

          Most spiders spin multiple types of silk, including silks for reproduction, prey capture, and draglines. Spiders are a megadiverse group and the majority of spider silks remain uncharacterized. For example, nothing is known about the silk molecules of Tengella perfuga, a spider that spins sheet webs lined with cribellar silk. Cribellar silk is a type of adhesive capture thread composed of numerous fibrils that originate from a specialized plate-like spinning organ called the cribellum. The predominant components of spider silks are spidroins, members of a protein family synthesized in silk glands. Here, we use silk gland RNA-Seq and cDNA libraries to infer T. perfuga silks at the protein level. We show that T. perfuga spiders express 13 silk transcripts representing at least five categories of spider silk proteins (spidroins). One category is a candidate for cribellar silk and is thus named cribellar spidroin (CrSp). Studies of ontogenetic changes in web construction and spigot morphology in T. perfuga have documented that after sexual maturation, T. perfuga females continue to make capture webs but males halt web maintenance and cease spinning cribellar silk. Consistent with these observations, our candidate CrSp was expressed only in females. The other four spidroin categories correspond to paralogs of aciniform, ampullate, pyriform, and tubuliform spidroins. These spidroins are associated with egg sac and web construction. Except for the tubuliform spidroin, the spidroins from T. perfuga contain novel combinations of amino acid sequence motifs that have not been observed before in these spidroin types. Characterization of T. perfuga silk genes, particularly CrSp, expand the diversity of the spidroin family and inspire new structure/function hypotheses.

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          Reconstructing web evolution and spider diversification in the molecular era.

          The evolutionary diversification of spiders is attributed to spectacular innovations in silk. Spiders are unique in synthesizing many different kinds of silk, and using silk for a variety of ecological functions throughout their lives, particularly to make prey-catching webs. Here, we construct a broad higher-level phylogeny of spiders combining molecular data with traditional morphological and behavioral characters. We use this phylogeny to test the hypothesis that the spider orb web evolved only once. We then examine spider diversification in relation to different web architectures and silk use. We find strong support for a single origin of orb webs, implying a major shift in the spinning of capture silk and repeated loss or transformation of orb webs. We show that abandonment of costly cribellate capture silk correlates with the 2 major diversification events in spiders (1). Replacement of cribellate silk by aqueous silk glue may explain the greater diversity of modern orb-weaving spiders (Araneoidea) compared with cribellate orb-weaving spiders (Deinopoidea) (2). Within the "RTA clade," which is the sister group to orb-weaving spiders and contains half of all spider diversity, >90% of species richness is associated with repeated loss of cribellate silk and abandonment of prey capture webs. Accompanying cribellum loss in both groups is a release from substrate-constrained webs, whether by aerially suspended webs, or by abandoning webs altogether. These behavioral shifts in silk and web production by spiders thus likely played a key role in the dramatic evolutionary success and ecological dominance of spiders as predators of insects.
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            Extreme diversity, conservation, and convergence of spider silk fibroin sequences.

            Spiders (Araneae) spin high-performance silks from liquid fibroin proteins. Fibroin sequences from basal spider lineages reveal mosaics of amino acid motifs that differ radically from previously described spider silk sequences. The silk fibers of Araneae are constructed from many protein designs. Yet, the repetitive sequences of fibroins from orb-weaving spiders have been maintained, presumably by stabilizing selection, over 125 million years of evolutionary history. The retention of these conserved motifs since the Mesozoic and their convergent evolution in other structural superproteins imply that these sequences are central to understanding the exceptional mechanical properties of orb weaver silks.
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              Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes

              Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: Data curationRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                20 September 2018
                2018
                : 13
                : 9
                : e0203563
                Affiliations
                [1 ] Department of Evolution, Ecology, and Organismal Biology, University of California, Riverside, California, United States of America
                [2 ] Department of Neurology, Oregon Health and Science University, Portland, Oregon, United States of America
                [3 ] J. Craig Venter Institute, Rockville, Maryland, United States of America
                [4 ] Division of Arthropods, Museum of Southwestern Biology, Albuquerque, New Mexico, United States of America
                [5 ] Division of Invertebrate Zoology and Sackler Institute for Comparative Genomics, American Museum of Natural History, New York, New York, United States of America
                Charles University, CZECH REPUBLIC
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-6889-2688
                Article
                PONE-D-18-06816
                10.1371/journal.pone.0203563
                6147414
                30235223
                af12b4ea-8679-4dcb-bc06-3eb069338214
                © 2018 Correa-Garhwal et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 March 2018
                : 27 July 2018
                Page count
                Figures: 2, Tables: 0, Pages: 14
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000183, Army Research Office;
                Award ID: W911NF-11-1-497 0299
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000183, Army Research Office;
                Award ID: W911NF-15-1-0099
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100007602, University of California, Riverside;
                Award Recipient :
                This work was supported by the Army Research Office (W911NF-11-1-497 0299 to C.Y.H. and W911NF-15-1-0099 to R.C.C. and C.Y.H.) and a Dissertation Year Program Fellowship from the University of California, Riverside Graduate Division to S.M.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Agriculture
                Animal Products
                Silk
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Arthropoda
                Arachnida
                Spiders
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Motif Analysis
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Arthropoda
                Arachnida
                Spiders
                Spider Webs
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
                Biology and Life Sciences
                Taxonomy
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
                Computer and Information Sciences
                Data Management
                Taxonomy
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
                Biology and Life Sciences
                Genetics
                Gene Expression
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Alignment
                Biology and life sciences
                Genetics
                DNA
                DNA libraries
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA libraries
                Custom metadata
                All sequencing data associated with this study has been deposited to NCBI. All raw sequencing reads are available in the NCBI Short Read Archive, accession number: SRR7186262 and SRR7186263. Tengella pergufa transcriptome is deposited in the NCBI Transcriptome Shotgun Assembly database (accession number GGOF00000000).

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